Axonal transport of dopamine-β-hydroxylase and acetylcholinesterase in human peripheral neuropathy

EXPERIMENTAL

66,467-478

NEUROLOGY

Axonal Transport cholinesterase

of Dopamine-P-hydroxylase and Acetylin Human Peripheral Neuropathy

STEPHEN BRIMIJOIN Departments

of

Pharmacology

Received

(1979)

February

AND PETERJAMES

and Neurology,

Mayo

21. 1979; revision

Clinic, received

DYCK'

Rochester, August

Minnesota

55901

3, 1979

The content and axonal transport of dopamine+-hydroxylase (DBH) were measured in biopsy samples of sural nerve from 20 normal subjects and from more than 50 patients with various types of peripheral neuropathy. In a smaller series of cases the dynamics of acetylcholinesterase (AChE) were also examined. Judged from the rate of accumulation of enzyme activity against a ligature during incubation in vitro, the normal average transport velocity of DBH was 1.8 mm/h. However, only 25% of the enzyme appeared to be transported and true velocity was estimated at 7.2 mm/h. Similar calculations placed the normal average velocity of AChE at 1 .Omm/h, the amount transported at 14%, and the true velocity at 7.1 mm/h. Statistically significant reductions in average transport velocity of DBH were observed in nerves from patients with hereditary sensory neuropathy types I and II and with hereditary motor and sensory neuropathy types II and III. In the latter condition (Dejerine-Sottas disease) transport was almost zero although the DBH activity per unit weight of nerve was normal. Significant reduction in average transport velocity of DBH but normal enzyme content was also observed in uremic neuropathy. In nerves from patients with diabetic neuropathy. both content and average velocity of DBH were reduced, and likewise those of AChE. Because the size of the transported fractions of these enzymes appeared normal in the diabetic nerves, the reduced average velocity probably reflected a real slowing of transport. These observations make it likely that rapid axonal transport of proteins is compromised in certain types of peripheral nerve disease in man.

INTRODUCTION The rapid bidirectional transport of organelles and macromolecules along the axons of peripheral neurons assures the delivery of synaptic Abbreviations: DBH-dopamine-P-hydroxylase, AChE-acetylcholinesterase. ’ We thank Ms. M. J. Wiermaa for technical assistance. This work was supported in part by center grants from the National Institutes of Health (NS 14304), the Muscular Dystrophy Association (12). and the Mayo Foundation. S.B. is a recipient of Research Career Development Award NSOOI 19 from the National Institutes of Health. 467

0014-4886/79/120467-12$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

468

BRIMIJOIN

AND DYCK

vesicles and enzymes to nerve terminals (9, 13), the supply of proteins to the axonal membrane (lo), and the delivery of neurotrophic factors to nerve cell bodies (23). The importance of these functions makes it logical to suggest defective axonal transport as a possible cause of peripheral nerve disease. This suggestion seems particularly appropriate for the socalled “dying-back” neuropathies, in which the distal parts of long neurons degenerate, as though in want of vital factors from proximal regions (8). Unfortunately, convincing evidence is lacking that defective transport is involved in dying-back neuropathies or any other disease of peripheral nerve. Although changes in rapid axonal transport were reported in animals with conditions such as hereditary murine dystrophy (1, 15, 16), experimental diabetes (22), and organic solvent intoxication (2, 17), the abnormalities were small and the relationship to human disease uncertain. Systematic studies of axonal transport in normal human nerves and in nerves of patients with peripheral neuropathy are clearly needed. Such studies are made possible by the marked independence of axons from cell bodies, which enables pieces of excised nerve to transport proteins normally for many hours in a suitable environment (20). By studying the redistribution of axonal enzymes in nerve samples isolated by double ligation, one can infer the approximate rates and amounts of transport. We applied such an approach to biopsy samples of human sural nerve incubated in vitro. Our previous studies were carried out with the enzyme dopamine+-hydroxylase (DBH), which is confined to adrenergic axons. The results showed that this enzyme was normally transported at an average velocity close to 2 mm/h, but scattered observations suggested that velocity might be much lower in certain types of peripheral nerve disease (5, 6). We now report measurements of the axonal transport of DBH in a larger population of normal human sural nerves and in nerves from an extensive series of patients with various inherited and acquired neuropathies. Special attention was given to diabetic neuropathy, in which the transport of the more widely distributed enzyme, acetylcholinesterase (AChE), was also compared with the normal case. The findings strongly support the view that major abnormalities of rapid axonal transport are associated with some diseases of peripheral nerve in man. METHODS

AND MATERIALS

Fascicular biopsy specimens of human sural nerve were obtained with informed consent from normal human volunteers and from patients with peripheral nerve disease. The advantages, limitations, and side effects of this procedure, which was performed in the operating room under local

TRANSPORT

AND

NEUROPATHY

469

anesthesia, were discussed previously in detail (12). Samples were collected in ice-cold Tyrode’s solution and brought to the laboratory within 15 min of removal. Under a dissection microscope they were trimmed carefully to remove adherent fat and connective tissue without disturbing the perineurium. A length of 3 to 6 cm with no fascicles entering or leaving was identified and ligated at both ends with silk thread (4-O gauge). The nerves were then transferred to incubation chambers, in which they were immersed in a bicarbonate-buffered physiological salt solution containing glucose, continuously bubbled with 95% 02-CO2 and kept at 37°C by a thermostatic circulator (3). Most incubations lasted 3 h; this period was chosen after experiments with normal nerves showed that enzyme activity accumulated steadily against the distal ligature for at least that long (5). After incubation the nerves were weighed and cut into consecutive 3-mm segments, which were individually homogenized in glass homogenizers each containing 0.6 ml of an icecold buffer: 0.05 M Tris-HCl,pH 7.4; 0.1% (v/v) Triton X-100; and 0.2% (w/v) bovine serum albumin. The homogenates were centrifuged 15 min at 1000 g and 4°C. Duplicate 200-~1 samples of the supernatant fractions were then assayed for DBH by the method of Molinoff et al. (18), using tyramine as a substrate and Ct.&O, to overcome endogenous inhibitors of the enzyme. DBH activity was expressed in units of picomoles octopamine produced per hour incubation. Duplicate 50-J samples of the supernatants were assayed for AChE by the method of Potter (19), using 14Clabeled acetylcholine iodide as a substrate and ethopropazine-HCl in a final concentration of 10e4 M to inhibit pseudocholinesterase. AChE activity was expressed in units of nanomoles acetylcholine hydrolyzed per hour incubation. Because total enzyme activity was previously found to be unaffected by incubation in vitro (5), changes in local activity were assumed to reflect redistribution of enzyme as a result of axonal transport. The accumulation of activity at the distal ligature was determined by subtracting the mean activity per segment from the activity in the segment next to the ligature. The average velocity of transport was then calculated as

A.L

v,, = -M.T 5 where V,, is in millimeters per hour, A is the accumulation, L is the length of each segment in millimeters, M is the average activity per segment, and T is the time of incubation in hours. Average velocity as so-derived provides a useful index of transport but is equal to the true velocity only ifall the enzyme in question is available for transport and is moving at the same rate.

470

BRIMIJOIN

AND

DYCK

In conditions where relatively large numbers of samples were available for study, an attempt was made to correct average velocity for the proportion of enzyme actually available for transport. For this purpose, the “clearance” of enzyme activity from the proximal part of each nerve was determined as follows. The segment next to the proximal ligature was excluded from the calculations because it was expected to accumulate enzyme undergoing retrograde axonal transport. Average enzyme activity in the next three segments was expressed as a fraction of the overall mean activity for an equivalent length of nerve. The difference from 1 .Oindicated the proportion of activity lost during incubation from the region in question and was defined as the clearance fraction. Assuming again that local changes in enzyme activity reflect redistribution as a result of transport and that 3 h of incubation is long enough for transported enzyme to leave the sampled region, this clearance fraction is an index of the proportion of enzyme available for axonal transport. One therefore calculates that V a” V true = 3 c where C is the clearance fraction. If the proportion of enzyme actually in motion at any given instant is not equal to the total proportion available for transport, then V,,,, will not be equal to the mean instantaneous velocity of transport. However, the value of this corrected velocity lies in the likelihood that it is independent of variations in the relative amounts of fixed and transportable material (4). RESULTS Axonal transport of DBH and AChE in vitro was measured in biopsy samples of nerve obtained during a 5-year period from normal human volunteers. The results, summarized in Table 1, revealed that the average transport velocity of the former enzyme was moderately but significantly greater than that of the latter. However, as the estimates of “clearance” showed, most of this difference was probably related to the relative sizes of the stationary and mobile fractions. The estimated velocities of mobile DBH and AChE were identical. Similar experiments were carried out on more than 60 biopsy samples from patients with various peripheral neuropathies and from some of these patients’ family members. In most cases only DBH was examined, but more recently AChE was examined as well. The results from each individual case for which a diagnosis was ultimately established are presented in the following tables, grouped by type of disorder according to the classification scheme of Dyck (11).

TRANSPORT

471

AND NEUROPATHY TABLE

1

Enzyme Content and Transport in Normal Sural Nerves

Enzyme Dopamine-P-hydroxylase (20 subjects. ages 20 to 50 years, mean 27) Acetylcholinesterase (12 subjects, ages 20 to 50 years, mean 30)

Content (unitsimg wet weight)

Average velocity” (mm/h)

Clearance fraction

True velocity (mm/h)

104 2 15

1.8 t 0.1

0.25

1.2

1.0 2 0.1

0.14

7.1

212

4

‘I Note that multiplying the content by average velocity yields a crude index of flux of enzyme activity in units/h/mm* of nerve, assuming that 1 mm nerve has a weight of 1 mg.

Hereditary neuropathies involving primarily one system are represented in Table 2. All values from cases of hereditary motor neuropathy were in the normal range. The V,,. for transport of DBH was significantly less than normal in the nerves from patients with hereditary sensory neuropathy type I. Although content of enzyme activity was also reduced in this condition, the differences from normal were not significant. In the two cases of hereditary sensory neuropathy type II, the mean value of V,,. for transport of DBH was more than two standard deviations less than normal, suggesting an abnormality of transport in this condition also. Hereditary neuropathies involving multiple systems are represented in Table 3. The V,, of DBH was significantly reduced in the nerves from patients with hereditary motor and sensory neuropathy type II, whereas content of enzyme activity was almost normal. Even more striking was the 90% reduction in V,, of DBH in the nerves from patients with hereditary motor and sensory neuropathy type III (Dejerine-Sottas disease). These remarkable abnormalities were accompanied by a normal content of DBH activity per unit weight, even though the samples weighed more than normal per unit length. In the single examined case of spinocerebellar degeneration, V,, for DBH was more than two standard deviations less than the normal mean, but the content of DBH activity was again normal. On the other hand, both V,, and content of DBH activity were far less than normal in the case of hereditary motor and sensory neuropathy type I. All measured values were normal in the single case of Shy -Drager syndrome. A few patients with hereditary neuropathy had relatives who consented to biopsy (Table 4). The only significant difference from normal was an elevation of V,, for DBH in nerves from the family of a patient with hereditary motor and sensory neuropathy type II. The four children of the patient with

472

BRIMIJOIN

AND DYCK

TABLE Dopamine+-Hydroxylase

Type of neuropathy Motor (peroneal muscular atrophy) Distal

2

Content and Transport in Hereditary Neuropathies Involving Primarily One System

Sensory HMSN I0

Mean k HMSN II

Average velocity (mm/h)

Sex

Age

11-73 53-7.5

M M

30 53

75 66 70

2.4 1.0 1.7

28-73

F

28

96

2.0

36-73 79-75 130-77 225-77

M F F M

38 13 67 54

16 78 31 133 64 k 26

0.0 1.7 1.0 1.7 1.1 k 0.4*

128-77 129-77

M M

14 23

77 72 74

0.1 0.6 0.4

Case

Mean Scapuloperoneal

Content (units/mg wet weight)

SE

Mean o Hereditary motor and sensory neuropathy type 1 * P < 0.05 vs. normal.

spinocerebellar degeneration were of special interest because they were at risk for inheriting the same disease by an autosomal dominant mechanism. Although three of the values for V,, lay at the lower limit of the normal range, there was no convincing evidence of abnormality in any sample. Relatively many samples were obtained from patients with diabetic neuropathy, and in most it was possible to assay AChE as well as DBH. Although DBH activity per unit weight was significantly reduced, there were still greater reductions in V,, (Table 5). Significant abnormalities were also found in the behavior of AChE in a largely overlapping series of samples (Table 6), but the reduction in content of this enzyme was more marked than the reduction in V,,. To determine whether or not the reduced V,, in diabetic nerves could have reflected a reduced proportion of moving enzyme as opposed to slowing of transport, the clearance of AChE and DBH was determined (see Methods and Materials). The values obtained, 14 and 22%, respectively, were almost equal to those from normal nerves (Table 1). The calculated

TRANSPORT

TABLE Dopamine-P-Hydroxylase

Type of neuropathy Motor and sensory HMSN I” HMSN II

3

Content and Transport in Hereditary Neuropathies Involving Multiple Systems Average velocity (mm/h)

Sex

Age

53-72

F

45

38

0.2

47-72 39-75 166-76 6-78

F M F F

50 62 19 15

96 43 44 Ill 14t 18

0.2 0.3 2.6 0.3 0.7 2 0.5**

161-72 47-11 48-77 160-77

F F M M

24 29 21 14

102 113 103 106 106 2 3

0.0 0.0 0.5 0.4 0.2 2 0.1*

5-73 34-11

M F

41 66

98 95

0.5 1.8

Mean + SE Central nervous system involvement Spinocerebellar degeneration Shy-Drager syndrome

Content (unitsimg wet weight)

Case

Mean k SE HMSN III

473

AND NEUROPATHY

u Hereditary motor and sensory neuropathy type 1. * P < 0.001 vs. normal. ** P i 0.01 vs. normal.

true velocity for transport of DBH in diabetic nerves was 0.610.22 = 2.7 mm/h, which like V,, was about one-third of the normal value. A similar calculation put the true velocity for transport of AChE in diabetic nerves at 1.0/0.14 = 3.6 mm/h, which like V,,. was one-half the normal value. Observations on the content and transport of DBH activity in samples from patients with miscellaneous acquired neuropathies are shown in Table 7. The statistically significant reduction in V,, for transport of DBH in uremic nerves is noteworthy because the content of enzyme activity was normal and these patients had few or no symptoms of neuropathy. Case 50-73 was biopsied on the contralateral side 7 months after the first sample was taken. During that interval, V,, increased substantially and enzyme content increased moderately. Unfortunately, although this patient had received regular dialysis, it was not ascertained whether improvements in his clinical condition correlated with the improvements in transport.

474

BRIMIJOIN

AND DYCK

TABLE Dopamine+-Hydroxylase

Kinship Propositus with HMSN II”

Mean +

Content and Transport in Relatives of Patients with Inherited Neuropathy

Propositus with spinocerebellar degeneration

Content (units/mg wet weight)

Average velocity (mm/h)

Case

Sex

Age

159-76 163-76 164-76

F F F

35 24 35

57 30 58 48k 9

3.6 2.5 2.4 2.8 2 0.4*

164-72

M

39

61

0.8

13-73 14-73 15-73 16-73

F F M M

16 17 15 13

138 112 66 92 102 * 15

1.0 1.2 2.0 1.1 1.3 + 0.2

SE

Propositus with HMSN III

Mean ?

4

SE

B Hereditary motor and sensory neuropathy type II. * P < 0.02 vs. normal.

DISCUSSION The present findings show clearly that reductions in the content and transport of enzymes accompany certain diseases of the peripheral nervous system. In the case of DBH such abnormalities point to involvement of the adrenergic nervous system. Abnormalities in the dynamics of AChE could reflect pathology in cholinergic nerves, but the wide distribution of this enzyme means that almost any kind of nerve could be involved. It is worth considering to what extent these abnormalities might reflect loss of axons in the samples studied or reduced synthesis and delivery of enzymes into them, as opposed to a derangement of the rapid transport system. First it should be noted that a loss of axons containing DBH or AChE could affect the measured content of enzyme. But a loss of axons could not change the calculated transport velocity, because the baseline enzyme activity per unit length of nerve would be reduced in parallel with the accumulation of activity against a ligature. One might also expect reduced enzyme content but normal calculated velocities in conditions leading to chronic reduction in the synthesis and delivery of enzyme into an otherwise normal population of axons. Either a loss of fibers or a reduction of synthesis could therefore account for the low content but near-normal velocity of DBH in the cases of chronic inflammatory polyneuritis, vasculitis, and pandysautonomia (Table 7).

TRANSPORT

TABLE Dopamine-P-Hydroxylase

Clinical status Polyneuropathy Polyradiculoneuropathy Polyradiculoneuropathy Polyneuropathy Polyradiculoneuropathy Polyneuropathy Polyneuropathy Polyneuropathy Polyneuropathy Polyneuropathy Mean t

SE

475

AND NEUROPATHY 5

Content and Transport in Diabetic Neuropathy

Case

Sex

Age

195-74 223-77 238-77 11-78 38-78 62-78 63-78 76-78 85-78 143-78

F M M F M F M F M M

74 50 52 66 67 55 32 23 60 69

Content (unitsimg wet weight) 49 42 146 45 65 36 28 68 57 29 56 -+ ll**

Average velocity Omnh) 1.0 0.9 0.7 0.8 0.2 0.6 0.2 0.4 1.3 0.0 0.6 + O.l*

* P < 0.001 vs. normal. ** P < 0.05 vs. normal.

Acute changes in the synthesis of DBH or AChE, however, could affect the calculated V,, of these enzymes by altering their distribution between moving and stationary phases in the axon (4). For this reason, an abnormal V,, does not prove that the transport system per se has been affected by a disease process. On the other hand, in many of the conditions studied here, such as the slowly developing hereditary neuropathies and other chronic illnessess, it seems unlikely that the rate of enzyme synthesis would change abruptly. Especially in cases like HMSN III (Dejerine - Sottas disease), in which enzyme content was normal although velocity was grossly reduced, major derangement of the transport system is highly probable. Diabetic nerves examined in the present system had considerably less than normal amounts of DBH and AChE activity per unit weight. Although unmyelinated axons were not counted, large reductions were found in the density of myelinated axons in these nerves (P. Dyck, unpublished observation), therefore reduced enzyme content may well reflect fiber loss as well as impaired protein synthesis. Because the same proportion of each enzyme was subject to clearance in the diabetic as in the normal nerves, however, there is reason to believe that the lowered V,, of these enzymes reflects a real slowing of transport. It would be desirable to confirm this conclusion by means of stop-flow experiments, which can provide direct measurements of transport velocity (3), and to combine this approach with

476

BRIMIJOIN

AND DYCK

TABLE Acetylcholinesterase

Clinical status Polyradiculoneuropathy Polyradiculoneuropathy Polyneuropathy Polyradiculoneuropathy Polyneuropathy Polyneuropathy Polyneuropathy Polyneuropathy Polyneuropathy Polyneuropathy Mean k

SE

6

Content and Transport in Diabetic Neuropathy

Case

Sex

Age

223-77 248-77 1 l-78 38-78 62-78 63-78 76-78 85-78 106-78 143-78

M F F M F M F M M M

50 67 66 67 55 32 23 60 57 69

Content (unitsimg wet weight)

Average velocity (mm/h)

7 13 19 1 4 4 7 6 3 11

0.4 0.5 0.1 0.1 0.4 1.3 0.9 0.9 0.0 0.0

7.5 k 1.7*

0.5 k 0.1**

* P < 0.001 vs. normal. ** P < 0.005 vs. normal.

teased-fiber analysis and electron microscope study of morphology in the sampled nerves. A final problem in interpreting the present observations concerns the role of microtubules, which are probably essential for rapid axonal transport (9, 13) but are depolymerized by cooling (7, 21). Because the biopsy samples of sural nerve were exposed briefly to low temperature after collection, they might have lost significant numbers of microtubules which would then have to repolymerize before transport could recover. Repolymerization of tubules and recovery of transport are known to be rapid in normal animal nerves (7) but could be slowed by peripheral nerve disease. Therefore it must be recognized that the abnormalities observed in axonal transport in sural nerve biopsy samples in v&-o might reflect some impairment in the recovery of transport in addition to or instead of preexisting deficiencies in the transport machinery. Further experiments will be needed to resolve this point. At present one cannot say whether or not reduced synthesis and transport of proteins causes any human neuropathy. It is quite possible that some of the changes described here are secondary to structural abnormalities such as axonal plugs of accumulated filaments or membranes (14, 17). Regardless of the origin, however, a block of transport could prevent materials essential for neurotransmission from reaching nerve terminals and could interfere with the repair and renewal of the axolemma,

TRANSPORT

TABLE Dopamine-/3-Hydroxylase

477

AND NEUROPATHY I

Content and Transport in Miscellaneous

Cause of neuropathy Uremia

Sodium cyanate

Average velocity (mm/h)

Sex

Age

185-72 9-73 50-73 50-73

M F M M

54 56 39 40

96 152 89 123 115 t 14

0.1 0.9 0.8 1.8 0.9 -+ 0.3*

158-72

F

66

37

0.3

70-73 71-73

F F

50 29

121 61 91

1.9 2.0 2.0

F

35

100

1.3

Mean Disulfiram

Content (units/mg wet weight)

Case

Mean + SE Carcinoma

Acquired Neuropathies

20577

Chronic inflammatory polyneuritis

71-75

M

44

21

1.4

Vasculitis

96-78

M

38

22

3.9

Pandysautonomia

28-77

F

54

29

1.2

138-73

M

58

94

2.1

Restless leg syndrome * P < 0.01 vs. normal.

thereby leading to failure of nerve function and even to neuronal degeneration. Abnormalities in transport therefore deserve further study as potential links in the development of peripheral nerve disease. REFERENCES W. G.. AND E. JAROS. 1973. Axoplasmic flow in axonal neuropathies. II. Axoplasmic flow in mice with motor neuron disease and muscular dystrophy. Brain 96: 247-258. BRADLEY, W. G., AND M. WILLIAMS. 1973. Axoplasmic flow in axonal neuropathies. I. Axoplasmic flow in cats with toxic neuropathies. Brain 96: 235-246. BRIMIJOIN, S. 1975. Stop-flow: a new technique for measuring axonal transport, and its application to the transport of dopamine-/3-hydroxylase. J. Neurobiol. 6: 379-394. BRIMIJOIN, S. 1976. Cycloheximide alters axonal transport and subcellular distribution of dopamine-p-hydroxylase activity. J. Neurochem. 26: 35-40. BRIMIJOIN, S., P. CAPEK, AND P. J. DYCK. 1973. Axonal transport of dopamine+% hydroxylase by human sural nerves in vitro. Science 180: 1295-1297. BRIMIJOIN, S., AND P. J. DYCK. 1974. Axonal transport of dopamine-P-hydroxylase by abnormal human sural nerves. Pages 291-294 in K. FUXE, L. OLSON, AND Y.

1. BRADLEY,

2. 3. 4. 5. 6.

BRIMIJOIN

478

7. 8. 9. 10. 11. 12. 13.

14.

15. 16. 17.

18. 19.

AND DYCK

ZOTTERMAN, Eds., Dynamics of Degeneration and Growth in Neurons. Pergamon, Oxford/New York. BRIMIJOIN, S., J. OLSEN, AND R. ROSENSON. 1979. Comparison of the temperature dependence of rapid axonal transport and microtubules in nerves of the rabbit and bullfrog. J. Physiol. (London) 287: 303-314. CAVANAGH, J. B. 1964. The significance of the “dying back” process in human and experimental neurological diseases. Inr. Rev. Exp. Pathol. 3: 219-267. DAHLSTROM, A. 1971. Axoplasmic transport (with particular respect to adrenergic neurons). Phil Trans. R. Sot. Lond. B. 261: 325-358. DROZ, B. 1975. Synthetic machinery and axoplasmic transport: maintenance of neuronal connectivity. Pages Ill-127 in R. 0. BRADY, Ed., The Nervous System. Vol. I, Raven Press, New York. DYCK, P. J. 1975. Inherited neuronal degeneration and atrophy affecting peripheral motor, sensory, and autonomic neurons. Pages 825-867 in P. J. DYCK, P. K. THOMAS, AND E. LAMBERT, Eds., Peripheral Neuropathy. Vol. 2. Saunders, Philadelphia. DYCK, P. J., AND E. P. LOFGREN. 1%8. Nerve biopsy: choice of nerve, method, symptoms and usefulness. Med. C/in. North Am. 52: 885-893. GRAFSTEIN, B. 1977. Axonal transport: the intracellular traffic of the neuron. Pages 691-717 in E. R. KANDEL, Ed., Handbook of Physiology, Section I: The Nervous System, Vol. I: Cellular Biology of Neurons. American Physiological Society, Bethesda, Md. GRIFFIN, J. W., AND D. L. PRICE. 1976. Axonal transport in motor neuron pathology. Pages 33-67 in J. M. ANDREWS, R. T. JOHNSON, AND M. A. B. BRAZIER, Eds., Amyotrophic Lateral Sclerosis, Recent Research Trends, UCLA Forum in Medical Sciences No. 19. Academic Press, New York. JABLECKI, C., AND S. BRIMIJOIN. 1974. Reduced axoplasmic transport of choline acetyltransferase activity in dystrophic mice. Nature (London) 250: 151-154. KOMIYA, Y., AND L. AUSTIN. 1974. Axoplasmic flow of protein in the sciatic nerve of normal and dystrophic mice. Exp. Neurol. 43: l- 12. MENDELL, J. R., Z. SAHENK, K. SAIDA, H. S. WEISS, R. SAVAGE, AND D. COURI. 1977. Alterations of fast axoplasmic transport in experimental methyl n-butyl ketone neuropathy. Brain Res. 133: 107- 118. MOLINOFF, P. B., R. WEINSHILBOUM, AND J. AXELROD. 1971. A sensitive enzymatic assay for dopamine-@-hydroxylase. J. Pharmacol. Exp. Ther. 178: 425-431. POTTER, L. T. 1%7. A radiometric microassay of acetylcholinesterase. .I. Pharmacol. Exp.

Ther.

156: 500-506.

20. OCHS, S., AND N. RANISH. 1969. Characteristics of the fast transport system in mammalian nerve fibers. I. Neurobiol. 1: 247-261. 21. RODRIGUEZ-ECHANDIA, E. L., AND R. S. PIEZZI. 1968. Microtubules in the nerve fibers of the toad Bufo arenarum Hensel. Effect of low temperature on the sciatic nerve. J. Cell Biol. 39: 491-497. 22. SCHMIDT, R. E., F. M. MATSCHINSKY, D. A. GODFREY, A. D. WILLIAMS, AND D. B. MCDOUGAL, JR. 1975. Fast and slow axoplasmic flow in sciatic nerve of diabetic rats. Diabetes 24: 1081- 1085. 23. STOECKEL, K., G. GUROFF, M. SCHWAB, AND H. THOENEN. 1976. The significance of retrograde axonal transport for the accumulation of systemically administered nerve growth factor (NGF) in the rat superior cervical ganglion. Brain Res. 109:271-284.